The fungal genus Cercospora includes several species that incite disease in economically important plants. For example, such species as C. arachidicola, C. zeae-maydis, and C. kikuchii are pathogenic to peanut, corn, and soybean, respectively. C. beticola causes sugar beet leaf spot resulting in considerable losses to the sugar beet industry of Europe and North America. This disease reduces tonnage, results in sugar beets with decreased sucrose content, and adversely affects the purity of the juice derived from infected plants. Cercospora species are unusual among plant pathogens due to their ability to attack a vast number and diversity of plant hosts. Additionally, a wide variety of organisms are sensitive to cercosporin including mice, bacteria, fungi and plants (5).
Cercospora species are aerial pathogens. Spores produced by these fungi germinate on the leaf surface and ultimately enter the leaf, e.g. through the stomata. Fungal mycelium then kills leaf cells and causes severe blighting of the leaf tissue by ramifying through the intercellular spaces in leaf tissues (5). Symptom development in infected plants is enhanced by high light intensities (3, 4), suggesting the involvement of light activation of the causative agent.
The phytotoxin cercosporin has been isolated from a number of Cercospora-infected plants and is believed to be a disease-inciting agent in these plants. See, e.g., 1, 7, 8. Previous methods of isolation have required several recrystallization steps to assure purity (see 1).
Cercosporin produced by C. beticolaand other species is a deep red pigment and has the molecular formula C.sub.29 H.sub.26 O.sub.10 and the structure depicted below (16). ##STR1##
The compound is a perylenequinone derivative with an unusual methylenedioxy-containing 7-membered ring. .sup.13 C-labeled sodium acetate precursors are incorporated in a pattern expected for a heptaketide, which subsequently dimerizes (11). When irradiated by light, the toxin produces singlet oxygen and superoxide, believed to cause peroxidation of membrane lipids Membrane damage leads to loss in membrane fluidity, leakage of nutrients and death of the plant cell (5).
Cercosporin is also known to be toxic to bacteria (9). The toxin inhibits growth in both gram-positive and gram-negative organisms, including members of the Bacillus, Clostridium, Pseudomonas and Staphylococcus genera, as well as inhibiting growth of E. coli (9). Bacteria and fungi vary in their sensitivity to cercosporin. In general the latter group of organisms are less sensitive than the former. Within the fungi, yeasts and several plant pathogens in the Ascomycete and Deuteromycete classes show resistance to the toxin while isolates of Neurospora crassa and several Aspergillus species, although taxonomically related, show sensitivity to the fungal toxin (5). The mechanism for resistance has not been elucidated.
It has not been possible to select for cercosporin-resistant plant cell mutants by mutagenesis and selection with cercosporin in plant tissue cultures (5). Therefore, the development of resistant plant varieties has been hampered. Thus, efforts to control cercosporin-producing fungi heretofore have focused on the application of fungicides, a practice which is costly, environmentally unsound, and has resulted in the development of fungicide-resistant pathogen strains.
It is now known that genes encoding desired proteins can be identified, isolated, cloned and expressed in transgenic organisms, including several important crop plants. One commonly used method of gene transfer in plants involves the use of a disarmed form of the Ti plasmid of the soil bacterium Agrobacterium tumefaciens (17). A. tumefaciens is a plant pathogen that causes crown-gall tumors in infected plants. Large plasmids, termed Ti- or tumor-inducing plasmids, are responsible for the oncogenicity of the bacterium as well as for the transfer of foreign DNA to the plant. Similarly, A. rhizogenes contains Ri- or root-inducing plasmids that induce root growth. Both plasmid types include a vir or virulence region that must be functional in order to transform wild-type cells to tumor cells (6).
Transformation results in the integration of another plasmid portion, termed the T- or transfer-DNA, into the nuclear genome of the transformed cells. Ri and Ti plasmids can be manipulated to allow insertion of foreign DNA, encoding a desired protein, into the T-DNA region. The foreign DNA can be transferred either via a vector bearing both the vir gene and the foreign gene or by a binary vector system consisting of two plasmids, one containing the vir gene and the other carrying the foreign gene. See, e.g., U.S. Pat. No. 4,658,082; Simpson et al. (13). Transformed plant cells can then be regenerated to produce varieties bearing the inserted gene. The production of transgenic, cercosporin-resistant plants will provide a useful and novel approach for the control of Cercospora-induced plant diseases.